The 11 surface immobilization antigens of stock 51 of the ciliate Paramecium tetraurelia are specified by a family of 11 unlinked genes, only one of which can be expressed at a time. Expression is determined by an interaction of cytoplasmic, genetic and environmental factors. We have devised a transformation system utilizing microinjection that makes it possible to introduce fragments of DNA containing these cloned genes into deletion mutants where they are telomerized, replicated and expressed in a normal fashion. We wish to continue our preliminary observations on copy number of these introduced fragments of DNA and investigate the existence of specific DNA sequences that are involved in replication of DNA in Paramecium. A second purpose of this project is to discover the molecular mechanisms involved in control of gene expression in the system. We plan to continue experiments designed to identify, isolate, and elucidate the functional regions of the genes involved in the expression of these antigens. Deletions produced in vitro, as well as chimaeras of genes that can be expressed at different temperatures will be used in transformation experiments to identify polymerase binding sites, sites where trans acting factors bind, and possible important regions within the coding sequences. We believe that the project will contribute to the general problems of gene replication and expression.